The long-term objectives of our research are to understand the organization and expression of regulatory and structural gene systems in simple eucaryotes, and to explain in molecular terms the function of the centromere in controlling chromosome segregation during mitotic and meiotic cell divisions. As a simple unicellular eukaryote, yeast is an excellent model system for studies on gene organization and regulation in higher cells. The methods we have developed over the past 3 years should now enable us to isolate and study any region of the yeast (or other simple eukaryote) genome, including centromeric DNA. Because of the lack of highly repetitive DNA sequences near the centromere of yeast chromosomes, we are able to isolate the centromeric DNA sequences by overlap hybridization techniques. Functional minichromosomes can be constructed, consisting of a yeast chromosomal replicator, a yeast centromere sequence, plus suitable genes for selection. These minichromosomes segregate as true chromosomes through mitosis and meiosis. Elucidation of the molecular events occurring during spindle formation and attachment of spindle fibers to chromosomes should now be possible. Current projects include: (a) determination of nucleotide sequence of yeast centromeres; (b) isolation and characterization of nuclear proteins binding specifically to centromeric DNA; (c) genetic studies on the behavior of minichromosomes in meiosis; and (d) functional and structural studies on yeast chromosomal replicators.